It is known to use immunodiagnostic procedures for the detection of antigens and antibodies in biological fluids. Such procedures have been developed for hormones, infectious agents, serum antibodies, pharmaceutical products, and drugs of abuse, to name a few. One procedure, enzyme-linked immunosorbent assay, or ELISA as it is sometimes called, has assumed a dominant role in diagnostic laboratories due to its relative sensitivity and specificity, while at the same time avoiding the need for extremely sophisticated equipment and radioactive materials. In the heterogeneous ELIZA procedure, an immunoreagent is immobilized on a carrier surface. The immunoreagent is capable of binding or reacting with the target material for which testing is being done. Following contact with the fluid suspected of containing the target material, a second, enzyme-conjugated reagent, incubated with the bound immunoreagent and washed to remove any unbound enzyme-conjugated immunoreagent. A substrate is then added which will develop a color reaction in the presence of the enzyme-conjugated immunoreagent. In a typical ELISA technique, a monoclonal antibody against a specific protein is absorbed to the surface of a plastic, e.g. polystyrene, microtiter plate well. The remaining surface binding sites are blocked, usually with glycine, avidin, or the like, to prevent nonspecific interaction at the surface of the plastic. The specific protein is then allowed to interact and bind to the monoclonal antibody by placing a fluid containing or suspected of containing the same in contact with the plastic surface. The surface is then washed to remove unbound protein. Enzyme-conjugated antibody is then added to the well and allowed to bind to the previously bound specific protein. The enzyme-conjugated antibody may be monoclonal, against the same or another epitope of the specific protein, or polyclonal against a number of epitopes of the specific protein. The microtiter well is then washed free of the unbound enzyme-conjugated antibody. A substrate capable of producing a detectable end product in the presence of the enzyme system is then added to the well and incubated. The presence of the end product is estimated visually or photometrically.
While the ELISA technique is advantageous in many respects as noted above, there remain numerous problems involved with its application. The immobilized antibody is typically bound to a bead or small particle, or coated on the surface of a microtiter plate well. This requires substantial incubation periods for each step in the procedure and considerable washing between steps. As a result, ELISA can be time-consuming. Even when several samples are run simultaneously, the procedure can require several hours to conduct the assay. In addition, there is a strict protocol for ELISA, particularly with respect to the timing of the incubation steps, and measuring of reagents, tending to limit the use of ELISA to large hospitals and clinical reference laboratories.
From U.S. Pat. No. 4,376,110, it is known to employ two-site immunometric assays using pairs of monoclonal antibodies, one bound to a solid phase, such as beads, and the other labeled in a solution phase to permit detection. The monoclonal antibody pairs recognize different epitopic sites on an antigen and obviate an intermediate washing step.
It is known from U.S. Pat. No. 4,446,232 to use a two-zoned device having bound antigens for enzyme-imunoassaying procedures. In this device, a top layer contains antibodies which react with antigens passing therethrough. A second layer contains immobilized antigens which will react with and adhere to, and thus immobilize, antibodies which have not reacted with the antigens in the first layer. A third layer containing a reactive substrate is positioned beneath the second layer and serves to detect through a color-forming reaction the antibody-antigen sandwiches which pass through the second or intermediate layer.
It is known from U.S. Pat. No. 4,632,901 to employ an apparatus including an immunoreagent-binding membrane adjacent to an absorbent material in conducting ELISA procedures. The absorbent material has capillaries formed by the orientation of fibers transversely to the membrane surface to draw immunoreagents, washing solutions and reactive substrates through the membrane.
ELISA procedures are described in detail in Voller et al., Manual of Clinical Laboratory Immunology, Chapter 17, pp. 99-109. Immunoblotting and slot-blot assay procedures using a membrane which covalently binds proteins are described in Towbin, Journal of Immunological Methods, Vol. 72, pp. 313-346 (1984); and Marlow et al., Journal of Immunological Methods, Vol. 101, pp. 133-139 (1987).
A method for the serological detection of antibodies to HTLV-III in sera of patients with AIDS and pre-AIDS conditions is described in U.S. Pat. No. 4,520,113.
Other references which describe assaying procedures include U.S. Pat. Nos. 3,962,413; 4,305,721; 2,370,683; and 4,078,892.